The general objective of these proposed studies is to understand how an organ is designed within the plant segment, and how regional competencies unfold over developmental time. Our system, the maize shoot segment and leaf organ development, now benefits from a skeletal network of function involving 10 cloned genes, at least eight of which are transcription factors. Some of these genes are essential for initializing particular domains of the segment. One is necessary to keep homeodomain genes "off' in the leaf. We hypothesize that each homeobox gene (knox) acts in varying regions of the segment to retard a "maturation schedule" of competence. Our proposal focuses on the axes of the leaf, and on whether or not our genes are involved in a long- range signaling pathway. Aim number l. How do Class I KNOX proteins function in the segment and leaf? Our strategy involves crossing our knox gene knockouts together to make minimized lines. These lines will be valuable for immunolocalization studies, assessing loss-of-KNOX- function phenotypes and for testing KNOX protein, trafficking. Rice will be used as a control genome. Aim number 2. We will determine if RS2 directly binds the rs1 gene. We will use a yeast one-hybrid screen and protein-DNA slot-blots. We will prepare an antibody against RS2 protein in order to test whether this Myb transcription factor binds knox DNA in vivo. Aim number 3. We will test further the maturation schedule hypothesis for regional fate acquisition and somatic inheritance along the p-d axis. We expect Knox function knockout phenotypes to accelerate development. Aim number 4. We will sequence ns genes and prepare an antibody to an NS protein, in collaboration. Aim number 5. We will use mosaic mapping to discover which, if either, epidermis is the "master" in switching ad-ab polarities, and will tag and clone the responsible gene, rld1. We intend to use the rld1 gene to further dissect the mechanism of initiation and maintenance of dorsiventrality. Aim number 6. We will confirm a clone of a gene involved in reception of long-range signals involving shoot developmental phase change (gef1), and will tag, clone and study lax midrib1, whose dominant allele Lxm1-O is a long-range accelerator of shoot development. We are elucidating a molecular genetic basis for heterochrony, the prevailing theory that links evolution with developmental biology.